Several concentrations of the PRO-EPI were tested against Gram-negative (E. coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus) microorganisms. The antimicrobial screening revealed that PRO-EPI was a potent antimicrobial agent with the least inhibitory concentrations (MICs) of 128 µg/mL against Gram-negative microorganisms. The PRO-EPI indicated no inhibitory effect against Gram-positive microorganisms. It was determined that PRO-EPI contains polymeric-quaternary ammonium compounds that inactivate the Gram-negative microorganisms by a dual mode of action and carries domains for electrostatic interaction with the microbial membrane and an intracellular target. To study the removal of toxic industrial wastewater, congo red (CR) was tested using sodium borohydride as a reducing agent. Adsorption was achieved within 20 min at a rate constant of 0.92 ks−1. UV–vis spectra showed that the removal of CR in the reaction solution was due to the breakup of the azo (–N=N–) bonds and adsorption of aromatic fragments. PRO is biodegradable and non-toxic, and PRO-EPI was found to be both antimicrobial and also acts as a catalyst for the removal of congo red dye.We report microbial resistance and catalytic activity of high viscosity cationic poly(proline-epichlorohydrin) composite (PRO-EPI) in the aqueous system. The PRO-EPI was prepared by a simple polycondensation, followed by FTIR,  Several chemicals such as iodophors, inorganic salts, organo-metallics, phenols, thiophenols, heterocyclics, urea, nitro compounds, and formaldehyde by-products are known for their antimicrobial activity. However, most of them are toxic and not readily biodegradable in the environment . VariousPositive charges of cationic polymers attached to surfaces of microbes were used as antimicrobial agents in sophisticated formulations or directly by themselves . Among tThe overarching goal of this study is to prepare a highly viscous crosslinked cationic PRO-EPI composite using the polycondensation technique while exploiting the antimicrobial and adsorptive properties. Proline (PRO) was chosen as one of the components for PRO-EPI because PRO-based cationic polymers are stable and biodegradable, have the ability to complex with DNA molecules, and are non-toxic in nature . The spe4), sulfuric acid (H2SO4) and Congo red dye (CR) were purchased from Sigma-Aldrich . Nutrient Agar was acquired from Oxoid LTD , and Fresh Mueller Hinton Broth was purchased from Sigma-Aldrich . Deionized water (≤18 MΩ) was used to prepare all aqueous solutions. The process for the synthesis of PRO-EPI was reported earlier [All chemicals were used as received. L-proline (PRO), epichlorohydrin (EPI), ethylenediamine (ED), sodium hydroxide (NaOH), hydrochloric acid (HCl), sodium borohydride (NaBH earlier .2SO4) was used to terminate the reaction. The details of the synthesis process are described elsewhere [In brief, polymerization was carried out in a three-necked round bottom flask containing a condenser and magnetic stirrer. Purified nitrogen was passed through the vessels for approximately 20 min. High viscosity crosslinked cationic PRO-EPI composite was prepared using polymerization of epichlorohydrin, followed by amination with proline and ethylenediamine. In brief, L-proline (3.55 g) along with distilled water and epichlorohydrin were mixed and stirred in the glass reactor at 25 °C, followed by the dropwise addition of ethylenediamine into the reactor. The temperature was then gradually increased to 70 °C, and the reaction was carried out for 1 h. The temperature of the mixture was further raised to 90 °C, and thereafter, an excess amount of epichlorohydrin was mixed, and the reaction was allowed to continue for a day in order to obtain highly viscous crosslinked composites. Concentrated sulfuric acid  was mixed with a stock solution of CR azo dye  in a quartz cuvette and PRO-EPI (25 µL) was added. The final volume of the mix was adjusted to 3 mL with DI water.The PRO-EPI was characterized by 1H-NMR (Bruker (400 MHz) spectrophotometer, Bruker, Billerica, MA, USA). The morphology of the PRO-EPI was analyzed by SEM . Functional groups of the PRO and PRO-EPI were studied using a Varian 800 FT-IR spectrophotometer . The viscosity of PRO-EPI was determined using Brookfield Viscometers . A Zeta-Sizer particle size  instrument was used to measure the zeta potential of the PRO-EPI. UV–vis spectrophotometry characterization of the PRO-EPI was carried out using a UV–vis spectrophotometer  with a scan range of 300–700 nm. Thermogravimetric analysis (TGA) of PRO-EPI was carried out using a TGA Q500 . The DSC/TGA study was conducted at a heating rate of 10 °C/min in the range of room temperature to 800 °C. The viscosity of the sample affects the microbial resistance of the polymer. A Brookfield Viscometer  was used to measure the viscosity measurement of the PRO-EPI at 750 rpm and 25 ± 0.05 °C. The experimental viscosity of the PRO-EPI was 940 cps. An aqueous solution of NaBHFresh Nutrient Agar media was prepared as follows: 28 g was weighed out into 3 separate one-liter glass bottles filled to the one-liter mark with distilled water and mixed until the powder completely dissolved. The bottles were sterilized by autoclaving for 15 min at 121 °C. The resultant agar was transferred into plates for solidification.Resazurin powder (0.2 g) was dissolved in 10 mL of distilled water (autoclaved), and the dye solution was rigorously mixed and instantly covered using an aluminum foil to avoid exposure to light ,42.S.aureus, E. coli, and P. aeruginosa were spread on nutrient agar slopes at 4 °C and subcultured onto blood agar plates for 24 h prior to usage.Mueller Hinton Broth was prepared following the manufacturer’s instructions. Nutrient Broth powder (23 g) was weighed into a 1 L glass bottle and filled with distilled water. The solution was mixed until the broth was completely dissolved. Followed by sterilization in Bijou bottles by autoclaving for 15 min at 121 °C. The microbial cultures of S. aureus (ATCC 29213) and Gram-negative (E. coli (ATCC 25922) and P. aeruginosa (ATCC 27853) bacteria. Test tubes were inoculated using a bacterial suspension (106 CFU/mL) and incubated at 37 °C for 24 h. The MICs were the least concentration of the extract, which did not result in any visible growth of bacteria in comparison to extract-free broths. Each extract was tested in triplicates (unless specified) with each bacterium. The activities of the synthesized PRO-EPI was examined using a Microplate Alamar Blue Assay (MABA) with 96-wells microplates, including a positive control that consisted of antibiotic and a growth control which was pure culture broth. Then 20 μL of different concentrations of PRO-EPI was added to two neighboring wells except for the positive and growth control sample wells. The total volume in each well reached 200 µL with the addition of 20 μL of Alamar Blue. The final concentrations of the examined PRO-EPIs were 256, 128, 64, 32, 16, 8, 4, 2, and 1 µg/mL. After incubation, data were recorded as MICs (minimum inhibitory concentrations) of each PRO-EPI that completely subdued the growth of microorganisms. PRO-EPI was assessed for antimicrobial properties against E. coli (ATCC 25922), P. aeruginosa (ATCC 27853), and S. aureus (ATCC 29213). Ciprofloxacin and Nalidixic were used as positive controls; an examination of the data showed that the polymer indicated no inhibitory properties against staphylococcus. However, PRO-EPI showed that the MICs of 128 µg/mL were effective against Gram-negative bacteria , 1740 cm−1 (C=O band stretching vibration), and 1631 cm−1 (υC=O band)  . A stron the PRO . The fol5.8 cm−1 . Weak vium group . Moreove1H-NMR spectrum for the PRO-EPI [ PRO-EPI  shows sh PRO-EPI . The sig PRO-EPI  connecte PRO-EPI , and the PRO-EPI .The morphology of the PRO-EPI surface was studied using SEM . SEM imaDSC/TGA data of the polymeric-quaternary ammonium salts (QAS) in the nitrogen atmosphere are shown in E. coli and P. aeruginosa are anionic microorganisms, electrostatic interaction plays a vital role in the adsorption process [4+, –NH4+) can be used to attach to the microorganism by an electrostatic interaction via covalent bonding. These ammonium functional groups escalate the cationic nature of PRO-EPI, thereby facilitating stronger microbe surface-binding capability, which effectively inhibits further growth of microorganisms. The first step of the CR degradation process involved the breakdown of the –N=N– bonds. Additionally, the band at 338.08 nm, which indicates the “benzene-like” structures of CR, was deconstructed to a certain extent during catalytic reaction [4 solution.PRO-EPI is composed of QACs that contain a positive charge in the backbone along with methyl pendant functional groups (hydrophobic segment). Several factors influence the activity, the most important being the cationic nature of the composite. This composite first adsorbs onto the membrane, then inserts the hydrophobic moieties into the lipid bilayer of the microbe, resulting in membrane structure damage ,50. It p process . The quaE. coli, P. aeruginosa, and S. aureus microorganisms. Their antimicrobial properties were compared with the positive control and growth control  present in PRO-EPI promoted the activity, which is further improved by the presence of hydroxyl (OH−) groups. The positively charged hydrogen on an –OH groups have the potential to attract the Gram-negative cell wall of the microbial membrane [E. coli and P. aeruginosa are negative, whereas PRO-EPI is positively charged; hence, it is expected that a much stronger surface-binding capability [S. aureus, disabling further cell growth. The MICs values of other commonly used pyridine-based and quaternary ammonium polymeric compounds of MICs were 200 and 59–156 µg/mL, respectively [MICs of both samples were 128 µg/mL for Gram-negative bacteria but not for Gram-positive bacteria, as observed by their activities. The higher activity of PRO-EPI at a MIC value of 128 µg/mL may be due to the presence of hydrophobic moieties such as methyl pendant reactive functional groups ,20. In amembrane . It may pability  results ectively ,55,56. M+ ions and other cytoplasmic constituents) [S. aureus was tested with the same MICs (128 µg/mL), there was no activity, indicating that PRO-EPI was effective only for the Gram-negative bacteria. This was expected, given the cell membrane is enclosed in a thick and porous cell wall that permits small molecules to diffuse. Since S. aureus contains thicker peptidoglycan, it was predicted that high viscosity/molecular weight polymers would not readily diffuse through this cell wall as in the case of the Gram-negative E. coli [S. aureus [S. aureus. On the other hand, the structure of S. aureus allows the PRO-EPI to collect at the outer and inner membranes. These findings could potentially explain why EPI-PRO inactivates E. coli and P. aeruginosa but not S. aureus.The activity increased as a result of the number of quaternized nitrogen atoms in the main chain. Moreover, the electrostatic interaction could result in adsorption of PRO-EPI onto the cell surfaces by and membrane damage may be caused by subsequent leakage of low molecular weight compounds (Kituents) ,58. In a E. coli , which h. aureus . The hig. aureus . Consequ. aureus  reported. aureus . The PROS. aureus bacterium [S. aureus strains may reduce attraction with the PRO-EPI antimicrobial agent, resulting in ineffective activity [S. aureus. This could be partially due to the reduced hydrophobicity and enhanced negative surface charge on Gram-negative microorganisms. It was observed that Gram-positive bacterium cells are in contact with cationic polymeric QACs. D-alanylation of teichoic acids and the lysylation of phosphatidylglycerol reduce the negative charge of the cell surface and membrane, thus resulting in hindering the adsorption of the cationic antimicrobials [Spacer length between the active sites within the polymer is noticeably different, and structure configuration also influences microbial activity. The long chain increases the spacer length and hindrance between the polymer and the hydrophobic bacteria . It is racterium , leadingacterium . Additioactivity . In summcrobials .4 and Azo dye was agitated, and the reaction was monitored using a UV-vis spectrophotometer (every two minutes) in the 300–700 nm range at room temperature  was determined from the linear plot of ln A vs. reduction time , which has lower toxicity [4 solution. The CR adsorption was very fast as monitored by UV-vis spectrometry.It should be noted that NaBHtoxicity . As descE. coli and P. aeruginosa degradation and simultaneous decolorization of CR. Electrostatic interactions played a major role in the adsorption process. MICs obtained in this study were much lower than the pyridine-based and quaternary ammonium polymeric compounds and also resulted in better antimicrobial activities. Negative bacteria showed high affinity and a higher level of susceptibility to PRO-EPI. The profile of the peaks co-evidenced in the UV-vis spectrometry confirmed the removal of CR. It was only necessary to employ 25 µL of PRO-EPI catalyst to achieve the adsorption of the azo compounds tested. Based on the results of this study, polyamine-based PRO-EPI could be an attractive catalyst to facilitate degradation in dye polluted industrial wastewater.Performance of PRO-EPI-based composites tested with two types of microorganisms under identical conditions was reported. PRO-EPI catalyst prepared using a simple polycondensation process was effectively applied for


Background. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) is the most transmissible ß-coronavirus in history, affecting all population groups. Immunocompromised patients, particularly cancer patients, have been highlighted as a reservoir to promote accumulation of viral mutations throughout persistent infection. Case presentation. We aimed to describe the clinical course and SARS-CoV-2 mutation profile for 102 days in an immunocompromised patient with non-Hodgkin’s lymphoma and COVID-19. We used RT-qPCR to quantify SARS-CoV-2 viral load over time and whole-virus genome sequencing to identify viral lineage and mutation profile. The patient presented with a persistent infection through 102 days while being treated with cytotoxic chemotherapy for non-Hodgkin’s lymphoma and received targeted therapy for COVID-19 with remdesivir and hyperimmune plasma. All sequenced samples belonged to the BA.1.1 lineage. We detected nine amino acid substitutions in five viral genes , grouped in two clusters: the first cluster with amino acid substitutions only detected on days 39 and 87 of sample collection, and the second cluster with amino acid substitutions only detected on day 95 of sample collection. The Spike gene remained unchanged in all samples. Viral load was dynamic but consistent with the disease flares. Conclusions. This report shows that the multiple mutations that occur in an immunocompromised patient with persistent COVID-19 could provide information regarding viral evolution and emergence of new SARS-CoV-2 variants. SARS-CoV-2 is the most transmissible ß-coronavirus in history . In compGenome sequencing, structural identification, and modifications in viral surface proteins (Spike) have been relevant for the design of effective vaccines and therapies . ImmunocIn this work, we describe the clinical follow-up and viral mutation profile in an immunocompromised patient with non-Hodgkin’s lymphoma over 102 days. The mutational signature found in this SARS-CoV-2 patient was similar to that reported in other publications. We found some biologically relevant mutations.Sample collection. Samples were collected with a flexible nylon swab to reach the nasopharynx. The swab was left in place for several seconds and slowly removed while rotating. The swab was then placed in 2 mL of sterile viral transport medium. SARS-CoV-2 RNA extraction and RT-qPCR detection. Total nucleic acid was extracted from 300 µL of viral transport medium from the NPSs or 300 µL of whole saliva using the MagMAX Viral/Pathogen Nucleic Acid Isolation Kit  and eluted into 50 µL of elution buffer. For SARS-CoV-2 RNA detection, 5 µL of RNA template was tested using TaqPath master mix . All tests were run on a Thermo Fisher ABI QuantStudio 5 real-time thermal cycler . Illumina Sequencing. The libraries were prepared using the Illumina COVID-seq kit, following the manufacturer’s instructions. First-strand synthesis was carried out with RNA samples. The synthesized cDNA was amplified using ARTIC primers V4, then was tagmented and adapted using IDT for the Illumina Nextera UD Indices Set A, B, C, D (384 indices) . Dual-indexed pair-end sequencing with a 150 bp read length was carried out on the NextSeq 2000 platform .Oxford Nanopore Sequencing. Libraries were prepared according to ARTIC Midnight protocol PCR tiling of SARS-CoV-2 virus with a rapid barcoding kit (SQK-RBK110.96) and sequenced on the GridION sequencing platform. We used the PCRT_9125_v110_revE_24Mar2021 protocol. A total of 800 ng of DNA library was loaded into a primed R.9 flow cell (FLO-MIN106). MinKNOW software v.21.11.7  was used to collect raw sequencing data. Oxford Nanopore Raw Data Processing and Sequencing Data Quality Assessment Basecalling and barcode demultiplexing were performed with EPI2ME Agent v3.5.4 using the Fastq QC+ARTIC+NextClade app.A 45-year-old patient with a history of non-Hodgkin’s lymphoma presented with recurrent symptomatic SARS-CoV-2 infection. Past medical history was unremarkable except for smoking since 16 years of age. He was referred to our hospital in April 2020 due to chronic cough, dyspnea, unintentional weight loss, and a mediastinal tumor; biopsy revealed a CD20+ diffuse large B-cell lymphoma. At the time of diagnosis, he had a massive left pleural effusion, for which an evacuating paracentesis was performed. He received six cycles of R-CHOP chemotherapy and 18 fractions of radiotherapy, achieving a partial remission. A second line of chemotherapy with R-ICE  was started in February 2021. In June 2021, he showed evidence of left chylothorax. From this moment on, the patient required the use of supplemental oxygen through a nasal cannula. In July 2021, central nervous infiltration was diagnosed, and he started chemotherapy with R-ESHAP  and intrathecal methotrexate as salvage therapy. He required chemical pleurodesis in August 2021. The last ESHAP was administered on 17 December 2021.On 29 January 2022, he was diagnosed with mild COVID-19 with a positive PCR test for SARS-CoV-2. He had not been vaccinated against SARS-CoV-2. At the time of diagnosis, he described mild respiratory symptoms without increased oxygen requirements. He evolved favorably without requiring further interventions. High-dose methotrexate was delayed until full symptomatic recovery and completion of the isolation period, and resumed thereafter. On the fourth day after chemotherapy infusion, he developed a low-grade fever and productive cough. Chest computed tomography (CT) revealed right parahilary condensation and air bronchogram. Antibiotic therapy with piperacillin/tazobactam was given for three days and subsequently switched to levofloxacin on an outpatient basis for five days. Three days later, he sought consultation due to persistent fever and was hospitalized for workup. The chest CT scan reported multifocal pneumonia and he was treated with piperacilin/tazobactam. Due to persistent fever, and new cutaneous lesions, a skin biopsy was performed with histopathology showing evidence of hematologic progression. On 3 March, he started fourth-line chemotherapy with bendamustin and brentuximab. As part of the workup and due to persistent fever, a new PCR test for SARS-CoV-2 was performed on 4 March, being positive. On 6 March, he received three days of remdesivir. On 8 March, one unit of 250 mL of hyperimmune convalescent plasma was infused. Fever resolved and he was discharged on 14 March with supplementary oxygen. His PCR was still positive on 11 March, with viral CTs ORF1a: 26.2, Spike: 0, and Nucleocapsid: 25.9.He received a second dose of chemotherapy on 1 April 2022, and on 13 April he was readmitted for fever and diarrhea, with no respiratory symptoms or changes in his chest CT. He was started on levofloxacin and piperacillin/tazobactam, with a poor response. On 20 April, his oxygen requirements increased, and chest CT reported new ground-glass opacities. The PCR for SARS-CoV-2 was positive, with viral CTs ORF1a: 15.4, Spike: 0, and Nucleocapsid: 14.2. He required non-invasive ventilation and was started on baricitinib and dexamethasone, with clinical improvement, with a new flare (fever and increased oxygen requirements) on 29 April, with a positive PCR with viral CTs ORF1a: 15.4, Spike: 0, and Nucleocapsid: 14.2. A new cycle of remdesivir was initiated for 5 days on 3 May with partial remission of the fever. On 6 May, his viral CTs were ORF1a: 26.8, Spike: 0, and Nucleocapsid: 25.3. On 13 May, the viral CTs were ORF1a: 34.2, Spike: 0, and Nucleocapsid: 32.8. A blood sample was analyzed for SARS-CoV-2-neutralizing antibodies on 13 May, between the first and the second convalescent plasma infusions, and there were no detectable antibodies. We were not able to measure SARS-CoV-2 viremia at any moment. He received a second infusion of hyperimmune convalescent plasma on 16 May. He continued with respiratory distress, but he did not accept invasive mechanical ventilation. Due to his unstable condition, no additional workup to discard other pulmonary conditions such as thromboembolism or opportunistic infections was performed. The patient died on 3 May 2022 .In relation to viral load, the patient presented two flares, both associated with chemotherapy. For the first flare on 3 March, there was clinical improvement with remdesivir and hyperimmune plasma, but viral load follow-up was not performed. For the second flare on 28 April, there was a decrease in the viral load after a new cycle of 5 days of remdesivir, plasma, and immunomodulatory therapy .All samples that underwent whole-virus genome sequencing belonged to clade 21K, lineage BA.1.1. Samples collected at days 1, 39, 87, and 95 had genome coverage >98% and the sample collected at day 102 had 90% coverage; interestingly, we observed dynamic behavior related to the presence and absence of amino acid substitutions through the days of collection in the N (nucleocapsid), ORF1a, ORF1b, ORF13a, and ORF9b genes. All analyses were focused on mutations that had a change in at least one sample collection point, indicating that they were not present at all collection times.We detected two clusters of amino acid substitutions. Cluster 1 showed a cluster of amino acid substitutions appearing on days 39 and 87, but absent on adjacent days 1, 95, and 102 (branch 1 of the dendrogram), and Cluster 2 was characterized by the presence of several substitutions at day 95 but absent on the other days (branch 2 of the dendrogram) A.In Cluster 1, we found four amino acid substitutions  detected only on days 39 and 87. The ORF1A:T1638I mutation was not detected on the first day of collection, but was detected on the rest of the days, unlike ORF1b:E1167V, which was only detected on the last day of collection. In Cluster 2, we found the presence of 4 mutations only on day 95 of collection . We did not find changes in the presence or absence of amino acid substitutions in the spike gene  throughoWe found that genomic profiles for samples collected on days 39 and 87 are nearly identical. Interestingly, the day 1 sample could be grouped either with the day 95 sample or with the day 102 sample, the last collection days. The ambiguous clustering of the sample from the last collection date (day 102) could be due to missing data, i.e., genomic positions that could not be called with high confidence . This mutation was previously observed in a patient with lymphoma in remission treated with rituximab and positive to SARS-CoV-2 for 161 days. At day 30 (second NPS collection), the virus showed a gain of the ORF1a:T1638I mutation, which remained for the next 161 days . LikewisThe mutation N:A119S is not present in variant BA.1.1, but in this report, was detected only on days 39 and 87; this amino acid substitution is present in 99.8% of cases of variant P.2, which circulated in Brazil in 2020 and, together with four other amino acid substitutions, evades neutralizing antibodies; the P.2 variant was classified as a variant of importance by WHO .The mutation ORF1b:P1427 has a deleterious effect on the protein ORF1b, implying changes in the fitness of SARS-CoV-2 variants harboring this mutation. This site may be a potential target for the development of therapeutic agents and deserves further functional studies . For theThe case reported highlights the complexity of treatment of COVID-19 in patients with hematological neoplasia. The patient described in this report showed the different biological scenarios in an individual with immunosuppression and prolonged viral shedding, and exemplifies a niche where the virus can evolve with the potential of mutation emergence and advanced SARS-CoV-2 fitness. Moreover, the description of the change in viral mutations during infection could provide information to learn more about the viral evolution and emergence of new variants .


Momordica charantia L. is a common edible medicinal plant that has various pharmacological activities and is often employed in daily health care. However, its anti-aging effect on mice and the underlying mechanism thereof remain unclear. Our current study mainly focused on the effect of Momordica charantia L. on d-galactose-induced subacute aging in mice and explored the underlying mechanism. UHPLC-Q-Exactive Orbitrap MS was applied to qualitatively analyze the chemical components of Momordica charantia L. ethanol extract (MCE). A subacute aging mice model induced by d-galactose  was established to investigate the anti-aging effect and potential mechanism of MCE. The learning and memory ability of aging mice was evaluated using behavioral tests. The biochemical parameters, including antioxidant enzyme activity and the accumulation of lipid peroxides in serum, were measured to explore the effect of MCE on the redox imbalance caused by aging. Pathological changes in the hippocampus were observed using hematoxylin and eosin (H&E) staining, and the levels of aging-related proteins in the PI3K/AKT signaling pathway were assessed using Western blotting. The experimental results demonstrated that a total of 14 triterpenoids were simultaneously identified in MCE. The behavioral assessments results showed that MCE can improve the learning and memory ability of subacute mice. The biochemical parameters determination results showed that MCE can improve the activity of antioxidant enzymes and decrease the accumulation of lipid peroxides in aging mice significantly. Furthermore, aging and injury in the hippocampus were ameliorated. Mechanistically, the results showed a significant upregulation in the protein expression of P-PI3K/PI3K and P-AKT/AKT (p < 0.01), as well as a significant reduction in cleaved caspase-3/caspase-3, Bax and P-mTOR/mTOR (p < 0.01). Our results confirm that MCE could restore the antioxidant status and improve cognitive impairment in aging mice, inhibit d-gal-induced apoptosis by regulating the PI3K/AKT signaling pathway, and rescue the impaired autophagy caused by mTOR overexpression, thereby exerting an anti-aging effect.Anti-aging is a challenging and necessary research topic.  Aging, from the beginning of life, is a complex, irreversible, multifactorial and inevitable physiological activity that everyone must experience. From a biological point of view, aging is the progressive degradation of physical compositions and tissue structures accompanied by a gradually loss of physiological functions. From a pathological viewpoint, aging is the accumulation of stress, injury, strain, immune decline, metabolic disorders and so on. It is continuous, causing a slowdown in metabolic rate, a decrease in body and organ function, and an increase in the prevalence of related diseases, including cardiovascular and cerebrovascular diseases, tumors and neurodegenerative diseases, making it considerably challenging to stay healthy . DelayinMomordica charantia L., commonly known as bitter melon, balsam pear and bitter gourd, is annual herbaceous climbing plant belonging to the family Cucurbitaceae. It originated in Asia but is more common in India and the south of China. It is widely planted in tropical and subtropical regions because of its high nutritional value and various pharmacological effects + ions were mainly generated, while in the negative ion mode, the compounds showed deprotonated [M − H]− ions and [M + HCOO]− ions. A preliminary analysis of the results showed that the chromatographic peaks generated in the negative mode were more abundant and the MS/MS compound fragment information was much clearer, which was beneficial to identifying the compounds. Therefore, the negative mode was chosen for the following process of analysis. The total ion chromatogram (TIC) of MCE  of MCE  showed tm/z values of the ions are expressed as integers to facilitate the statement in discussion. The glycosides of these triterpenoids are normally composed of β-d-glucopyranosyl (β-d-Glc), β-d-xylopyranosyl (β-d-Xyl), β-d-galactopyranosyl , α-L-rhamnopyranosyl (α-L-Rha), β-d-fucopyranosyl (β-d-Fuc) and β-d-allopyranosyl (β-d-All), or in the form of disaccharides or multiple saccharide chains, linked by O-bond substituents at different locations of aglycones. The characteristic neutral losses of these sugar residues are 132 Da (fructose), 146 Da (deoxyhexose) and 162 Da (hexose), respectively. The MS/MS spectra and MS extracted ion chromatogram (EIC) of goyasaponin II and momordicoside A are shown in − ion at m/z 1509 of goyasaponin II produces a fragment ion at m/z 1377 by the loss of α-L-Xyl residue (132 Da), an m/z 807 ion is produced by the loss of Xyl (1→3) Xyl (1→4) Rha (1→2) [Rha (1→3)] Fuc residue (132 Da + 132 Da + 146 Da + 146 Da + 146 Da), and an m/z 469 ion is produced by the loss of Gal (1→2) glucuronic acid residue on the basis of an m/z 807 ion. Momordicoside A is a tetracyclic triterpene of cucurbitane type with the molecular formula C42H72O15 and a molecular weight of 816. The [M − H]− ion of momordicoside A (m/z 815) at 9.20 min can be clearly observed in m/z 653 is produced by the loss of Glc residue (162 Da), and m/z 491 is generated by the loss of Glc (1→6) Glc (162 Da + 162 Da) residue. The information regarding the identification of the compounds is shown in Finally, a total of 14 major compounds, triterpenoids, were identified in MCE. The d-gal treatment resulted in a longer escape latency (p < 0.001), indicating that the mice developed severe cognitive impairment and that the model was successfully established . Moreover, the heat maps of spatial navigation, shown in d-gal group had a chaotic swimming route and most of them could not find the platform, while the mice in the MCE group could find the platform within a short distance and time. In a probe trial, the mice in the d-gal group mostly moved in the quadrant without a platform and spent a remarkably shorter time in the target quadrant compared with the control group (p < 0.001). Further, the platform crossing times of the d-gal group were notably fewer than those of the MCE group (p < 0.01), illustrated in Cognitive impairment along with a decline in learning and memory are symptoms of brain dysfunction that are closely related to aging. In a spatial navigation test, it was clear that long-term ablished . The micd-gal group shows a shorter escape latency and higher error times compared with the control group. As for the MCE group, the escape latency is longer (p < 0.001) and the number of errors is significantly lower than that in the d-gal group (p < 0.01). The above again indicates that a long-term injection of d-gal can damage the learning and memory ability of mice, establishing the success of the model. After training, the escape latency and error times of the step-down test in each group were calculated, and the results are shown in d-gal group decrease dramatically and are significantly heightened after MCE treatment (p < 0.01). As for the level of lipid peroxidation, malondiadehyde (MDA) and lipid peroxide (LPO) contents were measured. The MDA and LPO contents of the mice in the d-gal group show a significant increase and are both decreased by treatment with Vc and MCE with a significant downward trend (p < 0.01). The determination of these biochemical parameters indicates that long-term d-gal treatment resulted in severe oxidative damage in mice, while MCE can improve the oxidative stress of mice by increasing the activity of antioxidant enzymes to protect them from injury.The determination results of antioxidant enzyme activity in vivo are shown in d-gal group decreases significantly compared with the control group (p < 0.01), and this weight loss can be reversed by MCE. In addition, the spleen and thymus indexes of the d-gal group are also significantly lower than those in the control group . There are no adverse effects on organ index after treatment with Vc and MCE. As shown in d-gal group are disordered, most of which change from round to angular or other irregular shapes, accompanied by karyopyknosis, deepened staining and an apparent decrease in neuronal cell density. A large number of cell deletions and vacuole-like structures can be observed in the CA3 region. Additionally, most of the cells in the DG region are degenerated and the nuclei is pyknotic. Although there are elements of cell loss and hyperchromatic phenomenon in the three regions of the Vc and MCE group, there are no obvious abnormalities compared with the control group, which indicates that the degree of pathological changes in the hippocampus can be mitigated by MCE treatment.The hippocampus (HP), located in the temporal lobe of the brain, is part of the limbic system and plays a crucial role in learning, memory and spatial positioning. Cornu ammonis 1 (CA1), cornu ammonis 3 (CA3) and dentate gyrus (DG) are important regions of the HP with complex structures and are influenced by a variety of factors. Therefore, these regions were selected for comparison and analysis in this experiment. H&E staining of the CA1, CA3 and DG regions have been highlighted in d-gal administration produced a significant reduction in the protein expression of P-PI3K/PI3K and P-AKT/AKT (p < 0.01). Simultaneously, a marked elevation in the expression levels of cleaved caspase-3/caspase-3 (p < 0.001), Bax/β-actin (p < 0.001) and P-mTOR/mTOR (p < 0.01) are illustrated in d-gal in aging mice. In order to reveal the anti-aging mechanism of MCE, the expression of aging-related proteins in the hippocampus of subacute aging mice after MCE intervention were detected in this study. As shown in Momordica charantia L. is a common edible medicinal plant that contains various bioactive chemical components. Its chemical composition can be analyzed by nuclear magnetic resonance spectroscopy, but this requires high purity of the sample [e sample . In thise sample . By usinFinally, 14 triterpenoids were detected and identified in MCE. The saponins were proven to have the ability to prolong the lifespan of elegans and yeast, as previously described in the introduction. As a result, it is reasonable to consider that triterpenoids are the main anti-aging substances in MCE. Of course, it cannot be denied that other unidentified substances may also have anti-aging activities, and this requires further research.d-gal can weaken the activity of antioxidant enzymes and intensify lipid peroxidation in mice. Excessive free radicals can be eliminated by enhancing the activity of antioxidant enzymes or by supplementing exogenous antioxidants, so as to maintain the homeostasis of free radicals in the body and delay aging [d-gal-induced subacute aging in mice.The free radical theory and the oxidative stress theory are widely accepted in research on the aging process, and previous studies have shown that an excessive accumulation of free radicals is the cause of many diseases, including aging . Our resay aging . SOD, GSay aging . Our resay aging . In our d-gal exhibited significant memory loss, weight loss and an atrophy of the spleen and thymus indexes in our studies. The weight loss may be due to the reduction in usual exercise and food intake. Thymus is an important lymphatic organ in vivo and the site where T-lymphocytes differentiate, develop and mature. Meanwhile, the spleen, the largest peripheral immune organ, is the main site of specific immunity. Spleen and thymus indexes are indicators to evaluate the development of immune organs in the body [Further, it has been demonstrated that mice with an intraperitoneal administration of the body . Aging bthe body . Accordid-gal group. However, MCE can reduce the apoptosis of hippocampal neurons as well as maintain the normal morphology and function of the hippocampus.In order to examine the effects of MCE on aging mice from different perspectives, pathological observations of the hippocampus were also carried out. Previous studies have shown that the number and density of neurons in the hippocampus are inversely correlated with age . In the d-gal [d-gal treatment, while MCE could reverse this situation, MCE can directly or indirectly activate AKT by activating PI3K, with the activated AKT achieving a negative regulation of proapoptotic protein Bax and caspase 3, thus promoting cell proliferation, survival and a reduction in apoptosis. Therefore, we concluded that the anti-aging effect of MCE might be achieved by activating the PI3K/AKT signaling pathway and inhibiting the expression of Bax and caspase-3.Previous studies have shown that apoptosis contributes to the aging process induced by d-gal . Apoptosd-gal . PI3K/AKd-gal . AKT, acd-gal . Bax, cad-gal . Among td-gal ,41. Howed-gal treatment by reducing the level of P-mTOR/mTOR, thereby promoting cell self-renewal in aging mice.mTOR belongs to the PIKK family and is closely related to protein synthesis and autophagy in the body. mTOR promotes protein synthesis when it is highly expressed in an appropriate range but inhibits autophagy when over-regulated ,43. ApopMomordica charantia L. was obtained from Shouguang country , identified by Dr. Bo Li  and stored in the Jilin Ginseng Academy with voucher specimen No. 20200016. Both the HPLC-grade acetonitrile (ACN) and formic acid were purchased from Fisher Scientific . The Milli-Q® IQ 7000 system  was used to purify 18 MΩ water. d-gal (99% purify) and Vc were purchased from Sigma Chemical Co. . Ethanol and other chemicals were of analytical grade. Commercial kits for the measurement of CAT, SOD, GSH-Px, MDA and protein content  were obtained from Nanjing Jiancheng Biotech. Co., Ltd. . An ELISA kit for the determination of lipid peroxides (LPO) was purchased from Yi Feixue Biotech. Co., Ltd. . Polyvinylidene fluoride (PVDF) membranes (0.45 μm), RIPA, cocktail, PMSF, phosphorylase buffer, protein loading buffer and quick blocking buffer were obtained from Servicebio Co., Ltd. . PI3K, P-PI3K (Tyr458-199), AKT, P-AKT (Ser 473), caspase-3, cleaved caspase-3, Bax, mTOR, P-mTOR (Ser 2448), β-actin antibodies (rabbit anti-mouse) and horseradish peroxidase-conjugated antibody (goat anti-rabbit) were acquired from Affinity Biosciences Co., Ltd. . ECL chemiluminescent substrate was obtained from New Cell & Molecular Biotech. Co., Ltd. .Fresh v/v) and extracted in an ultrasonic bath at 45 °C for 30 min. After repeating the extraction 3 times, the 3 aliquots were combined and centrifuged at 10,000 rpm for 10 min, then the MCE was prepared. Part of the MCE was filtered through a 0.45 μm filter for analysis of UHPLC-MS, while the other was evaporated under nitrogen and redissolved with water for intragastric administration in mice.In this experiment, the fruit part of the plant was selected. The pulp and seeds were removed after washing, then the fruit was cut into 1 cm thick slices and placed in an oven at 105 °C for 20 min first, then transferred to an oven at 45 °C for drying to obtain dried samples. Soon afterwards, the samples were ground into powder, then immersed in 70% aqueous ethanol  and mobile phase B  were used with the flow rate of 0.3 mL/min. The liner gradient elution was carried out as follows: 0–2 min (5–20% B), 2–8 min (20–40% B), 8–15 min (40–45% B), 15–25 min (45–60% B), 25–28 min (60–95% B) and 28–30 min (95–5% B). The injection volume was 3.0 µL, and samples were kept at the temperature of 4 °C.UHPLC analysis was performed on an Ultimate 3000 high-performance liquid chromatography system equipped with a PDA detector and a hybrid quadrupole high-resolution accurate mass (HRAM) Orbitrap  system. The instrument was calibrated to reduce errors by using a calibration solution. A reversed-phase BEH RP-18 column  balanced at 45 °C was used for chromatographic separation. Mobile phase A  and analyzed by Xcalibur 4.1 software .MS spectrometric detection was performed on a Q-Exactive Orbitrap MS  equipped with an electrospray ionization (ESI) source. The ESI source was configured as follows: the full scan and ddMS2 mode in both positive and negative ion modes were selected in this experiment over an n = 20): the control group , the d-gal group , the Vc group  and the MCE group ; the volume of gavage in the control and d-gal groups was equivalent. The whole experiment lasted 6 weeks.Eight-week-old male Kunming mice (weight: 20 ± 2 g) were obtained from Changsheng Laboratory Animal Technology Co., Ltd., Liaoning, China (permit number: SCXK2020-0001). The animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Changchun University of Chinese Medicine (permit number: CPCCUCM IACUC 2021-062). After a 1-week acclimation to the laboratory environment , the mice were divided into four groups randomly  in the serum samples was carried out according to the manufacturer’s protocol. The spleens and thymuses were obtained for the calculation of organ index (organ weight/body weight (mg/g)) . SimultaThe fixed hippocampi were observed by H&E staining . BrieflyThe appropriate hippocampi were weighed and immersed in a complete RIPA (RIPA:Cocktail:PMSF:Phosphorylase inhibitor = 100:2:1:1) buffer, then homogenized with a low-temperature grinding instrument, followed by an ice bath for 30 min to lyse completely. Later, centrifuged at 10,000 rpm for 10 min, the supernatant was collected for protein content determination using a BCA protein assay kit. Finally, a protein loading buffer was added, and the samples were boiled for 10 min. An amount of 10 μL of the sample (20 μg protein) was separated on 6–10% sodium dodecyl sulfate (SDS)-polyacrylamide gels , electrophoresed and transferred to PVDF membranes. The membranes were incubated with PI3K, P-PI3K, AKT, P-AKT, caspase-3, Bax, mTOR, P-mTOR and β-actin antibodies at 4 °C for 24 h after quick blocking. Then, a horseradish peroxidase-conjugated secondary antibody was added to incubate for 1 h at an ambient temperature with shaking. Finally, the membranes were developed with an ECL kit, visualized and captured by an imaging system . Image J software was used to analyze the gray value of the target proteins.t-test, while a one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test, was used to compare multiple groups. It was considered statistically significant when p < 0.05.All data were presented as mean ± S.D. and analyzed using GraphPad PRISM version 8.0.2 software . The differences between the two groups were determined using Student’s Momordica charantia L. on subacute aging mice. The study of the chemical components of Momordica charantia L. indicated that the anti-aging effect may be closely related to triterpenoids. The biochemical and pharmacological parameters indicated that Momordica charantia L. could maintain redox balance and reduce oxidative stress in aging mice. It has the potential to promote cell survival and to reverse the decline in learning and memory ability caused by d-gal-induced subacute aging in mice by activating the PI3K/AKT signaling pathway. Furthermore, protective autophagy was activated by inhibiting the overactivation of mTOR. As a result, Momordica charantia L. may be an efficient treatment in aging, which warrants further research.This experiment was the first to demonstrate the anti-aging effect of


The blood–brain barrier (BBB), which is comprised of brain capillary endothelial cells, plays a pivotal role in the transport of drugs from the blood to the brain. Therefore, an analysis of proteins in the endothelial cells, such as transporters and tight junction proteins, which contribute to BBB function, is important for the development of therapeutics for the treatment of brain diseases. However, gene transfection into the vascular endothelial cells of the BBB is fraught with difficulties, even in vitro. We report herein on the development of lipid nanoparticles (LNPs), in which mRNA is encapsulated in a nano-sized capsule composed of a pH-activated and reductive environment-responsive lipid-like material . We evaluated the efficiency of mRNA delivery into non-polarized human brain capillary endothelial cells, hCMEC/D3 cells. The ssPalm LNPs permitted marker genes (GFP) to be transferred into nearly 100% of the cells, with low toxicity in higher concentration. A proteomic analysis indicated that the ssPalm-LNP had less effect on global cell signaling pathways than a Lipofectamine MessengerMAX/GFP-encoding mRNA complex (LFN), a commercially available transfection reagent, even at higher mRNA concentrations. Various cells are currently available as in vitro models for the BBB model. However, none of them completely reflect in vivo brain capillary endothelial cells. One example, a human brain capillary endothelial cell line hCMEC/D3), has a smaller claudin-5 expression than isolated brain capillaries and therefore results in the formulation of weaker tight junctions . Another, has a sKa) of around 6.3. Therefore, under physiological conditions, the ssPalm-LNP would behave as a neutral nanoparticle. However, in the acidic environment of endosomes, they would become positively charged and then fuse with the negatively charged cell membrane. The fusion between LNP and cell membrane in acidic endosome led to membrane disruption and subsequent endosomal escape of LNP to cytosol. The ssPalm also reacts with reducing agents such as the glutathione present in the cell and disintegrates, thus releasing the loaded nucleic acid into the cytoplasm [To achieve a more efficient gene delivery into human brain capillary endothelial cells, hCMEC/D3, we developed lipid nanoparticles (LNPs) that contain encapsulated mRNA . This LNytoplasm . Controlytoplasm . In thisRegarding the evaluation of adverse effects after the transfection, a comprehensive analysis of the protein expression level would be useful. The SWATH-MS  method is one of the recently developed quantitative proteomics methods, whose quantitative accuracy is higher than previous comprehensive proteomics technology . Using tBased on these analyses, we report herein on the advantage of using the ssPalm-LNP in terms of achieving a high mRNA transfection to the non-polarized human brain capillary cells, hCMEC/D3, with low adverse effects.2 atmosphere for 3–4 days, and maintained until used for mRNA transfection.The detail in the hCMEC/D3 culture procedure was almost the same as previously reported but slightly modified ,10,11. Tsn-glycerophosphocholine , cholesterol  and poly(ethylene) glycol (average molecular weight 2000)-1,2-dimyrisotyl-sn-gycerol  was prepared as previously [2+ and Ca2+ (PBS). The obtained LNP was characterized using a ZetaSizer Pro . The recovery rate and the encapsulation efficiency of the mRNA were determined by a RiboGreen assay, as previously reported [The GFP-encoding mRNA modified with 5-methoxyuridine was obtained from TriLink Biotech . The ssPalm-LNP using SS-OP (provided by NOF CORPORATION), 1,2-dioleoyol-eviously ,13. The eviously . Brieflyreported ,13. To lLipofectamine MessengerMAX  was used as a control. According to the manufacturers’ protocol, 1.5 μL of Lipofectamine Messenger MAX was incubated with 1000 ng of GFP-encoding mRNA for 10 min.4 cells/well 24 h before the addition of the ssPalm LNP and LFN. The cells were incubated with the DiD-labeled ssPalm-LNP at a dose of 300 ng mRNA in 750 μL of culture medium with 5% fetal bovine serum (0.4 μg/mL) for 2 h (cellular uptake) at 37 °C. To prepare the DiD-labeled LFN, DiD was first mixed with the Lipofectamine MessengerMAX solution so that the final fluorescence intensity in the LFN solution was the same as that for the ssPalm-LNP solution.hCMEC/D3 cells were plated on a 12-well plate at a density of 7.5 × 10To measure GFP expression, the cells were exposed to the ssPalm-LNP solution that had been diluted with a culture medium at an mRNA concentration of 300 ng in 750 μL (0.4 μg/mL) for 16 h. The dilution volume of the ssPalm-LNP was adjusted based on the mRNA recovery ratio measured by the RiboGreen assay result in each experiment. As a control, the LFN was then added to cells cultured in a 12-well plate and incubated for 16 h. The incubated cells were then washed twice with 1.0 mL of PBS and then trypsinized. The obtained cells were suspended in PBS with 0.5% bovine serum albumin and 0.1% sodium azide and analyzed using a NovoCyte .hCMEC/D3 cells were plated onto a glass-based 8-well chamber plate, which had been pre-coated with type-I collagen, at a density of 500 cells/well 48 h before the addition of the ssPalm-LNP or LFN. The cells were incubated with the ssPalm-LNP at a concentration of 300 ng/200 μL (0.4 μg/mL) for 48 h at 37 °C in the presence of 5% fetal bovine serum. As a control treatment, cells were exposed to LFN at the same dose as the ssPalm LNP. Both the ssPalm-LNP and the LFN complex were first diluted in 200 μL of culture medium. Nuclei were stained by a 10-min incubation in 1.0 μg/mL Hoechst33342. After washing with 200 μL of PBS, cells were observed by a Nikon C1 confocal laser scanning microscope system .2 in the presence of 5% fetal bovine serum, and observed under a microscope.The hCMEC/D3 cells were treated with the ssPalm-LNP containing GFP-encoding mRNA, and the LFN with 1250, 2500, and 6250 ng of GFP-mRNA in 1 mL  for 48 h at 37 °C in 5% CO2. The SWATH-MS analysis was then performed for the whole cell lysate of hCMEC/D3 cells, as previously described [p values were calculated, and based on a cutoff of 0.05, the issue of whether there is a significant difference in protein expression level between the groups was determined.The ssPalm-LNP and the LFN were treated with hCMEC/D3 cells at a concentration of 2.5 μg/mL for 48 h at 37 °C in 5% COescribed ,16. Brieescribed ,18. FinaThe ssPalm-LNPs that were manually prepared by the vortex method had an average particle size of 106.0 ± 15.0 nm, and polydispersity index was 0.09 ± 0.02 in PBS. The ζ-potential of −1.5 ± 1.0 mV in 10 mM HEPES buffer (pH 7.4). The distribution in particle size is shown in GFP expression was initially observed by microscopy . Most ofCellular uptake was also evaluated to determine the reasons for the ssPalm-LNPs being highly expressed in hCMEC/D3 cells. We found that the more homogenous the particles A, the hiTo observe the cytotoxicity of the ssPalm-LNP and LFN, hCMEC/D3 cells were observed after treatment at a higher concentration than the above concentrations that were used in these gene expression experiments. In the LFN-treated group, some cells were detached at higher concentrations . The morTo evaluate possible effects on off-target proteins, hCMEC/D3 cells were analyzed after being exposed to a higher concentration of the ssPalm-LNP and LFN than used in the gene transfection experiment. A total of 1899 proteins were quantified by SWATH-MS analysis in the control, ssPalm-LNP-, and LFN-treated groups . The levIn the above experiments, unlike LFN, the ssPalm-LNPs did not cause cytotoxicity. To understand the molecular mechanisms that are involved in this difference, proteins with significant differences in expression between the LFN and control groups, but not between the ssPalm and control groups, were extracted. As a result, 67 proteins were extracted . These wIn the present study, we reported that mRNA-loaded ssPalm-LNPs can be used to efficiently transfect hCMEC/D3 cells with a marker gene GFP without any obvious toxicity. Although previous reports have not provided a clear reason for the low transfection efficiency in BBB-derived endothelial cells, the present results suggest that one major factor is the low uptake of cationic substrates . CommonlThe ssPalm-LNPs did not alter the morphology of the hCMEC/D3 cells , even thWhen misfolded proteins accumulate in the cell, the unfolded protein response (UPR) is activated at the endoplasmic reticulum (ER) to reduce the accumulation of misfolded proteins. If the UPR fails to restore the ER to normality, ER stress can promote apoptosis . In HUVEThe currently reported mRNA transfection technology could be applied to the expression of various genes. We demonstrated the successful encapsulation of mRNA molecules ranging from 850 bases to more than 4.5 kilobases regardless of the presence or absence of chemical modification  . AdditioWe report herein that the ssPalm-LNP is a promising carrier for efficiently transporting mRNA into the non-polarized human brain capillary endothelial cells, hCMEC/D3. The transfection efficiency of the ssPalm LNP was found to be much higher than that of a commercially available transfection reagent Lipofectamine MessengerMAX. Treatment with high concentrations of the ssPalm-LNP did not induce cytotoxicity in comparison with LFN. Further, a SWATH-MS analysis revealed that the exposure of hCMEC/D3 cells to the ssPalm-LNP had only minimal effects on cellular proteins. These results indicate that the ssPalm-LNP represents a potent tool for elucidating the functional, biological chrematistics of the brain endothelium by the transfection of a gene of interest.


Being the most common cause of implant failure, peri-implantitis is defined as a pathological condition associated with the occurrence of peri-implant plaque, characterized by peri-implant mucosal inflammation and progressive loss of the supporting bone tissue attributed to the persistence of pro-inflammatory cytokines. Docosahexaenoic acid (DHA), which is a type of omega-3 polyunsaturated fatty acid, is generally used for the treatment of many inflammatory diseases. However, a suitable form for dosing and its therapeutic effect on peri-implantitis remain unclear. In this study, a novel nanostructured lipid carrier (NLC) loaded with squalene and DHA was fabricated (DHA-loaded NLC). The encapsulation efficiency and drug loading efficiency values of the DHA-loaded NLC were 78.13% ± 1.85% and 28.09% ± 0.48%, respectively. The release of DHA was gradual and steady until 144 h. In addition, the free-radical-scavenging rate of DHA-loaded NLC (0.57 ± 0.03) was much higher than that of sole DHA (0.17 ± 0.003). By inhibiting nuclear factor-κB p65 nuclear translocation, DHA-loaded NLC prevented the activation of nuclear factor-κB downstream inflammatory pathways and exerted anti-inflammatory effects on macrophages. Moreover, DHA-loaded NLC showed better effects on preventing alveolar bone resorption of rat peri-implantitis model than sole DHA. Hence, DHA-loaded NLC enhanced the anti-inflammatory bioavailability of DHA, offering a novel approach for the treatment of peri-implantitis. Due to the popularity of implant restorations, peri-implantitis has become a major postoperative complication , with prOmega-3 polyunsaturated fatty acid (PUFA), an essential PUFA found in large amounts in cod liver oil and seaweed , is wideNano drug-delivery systems, such as nanoemulsions, are effective ways to protect, transport, and address bioactivity and insolubility . A novelIn this study, squalene was used as liquid lipid, and the required DHA-loaded NLC was prepared by the phacoemulsification method to determine its anti-inflammatory effect and mechanism in LPS-stimulated macrophage inflammation and rat peri-implant inflammation models . The nulIn this study, a homogeneous nanoemulsion A with av−1 and C=H at 3013 cm−1  staining proved that there was almost no inflammatory tissue in the control group. In contrast, peri-implantitis lesions in the LPS group showed the most pronounced inflammation, manifested as higher densities, numbers, and area proportions of plasma cells, macrophages, as well as neutrophils. Meanwhile, inflammatory cell infiltration throw was relatively obvious in the NLC group, while the DHA and DHA-NLC groups showed a significant reduction and basic disappearance of inflammation in the peri-implant tissues. D.The micro-CT results are shown in The rats in the experimental group continuously increased in weight throughout the experiment, except for the initial stages of implantation and ligation A. In addIn order to validate the role of DHA in peri-implantitis and to improve its bioavailability, a nanostructured lipid carrier with DHA encapsulated using squalene as a liquid lipid was developed in this study. The common preparation methods of NLC include high-pressure homogenization, microemulsion, the solvent injection method, phase inversion, and ultrasonic emulsification . The ultP. gingivalis LPS at 5 μg/mL. The pro-inflammatory response was suppressed to varying degrees in each experimental group when the dose was 50 μg/mL, according to the qRT-PCR data. Additionally, this concentration was inside the drug’s acceptable limit. In comparison to the DHA and NLC groups, DHA-loaded NLC had a larger impact on the decreases in IL-1β, IL-6, and TNF-α. Previous animal studies demonstrated that wrapping a ligature around the implant neck in a submucosal position accelerated plaque formation, leading to the development of peri-implantitis lesions, similar to the development of natural diseases [In this experiment, a series of studies was carried out to verify the effect of DHA-loaded NLC on peri-implantitis in vitro and in vivo. RAW 264.7 macrophages dramatically increased their expression of inflammatory factors when exposed to diseases . In anotdiseases . TherefoContinuous inflammation, which results from an imbalance in the immune system’s control, is key to the degeneration of the peri-implant tissue. The development and progression of peri-implantitis are significantly influenced by different immune cells, as well as the precise expression of different immune cells and cytokines . As a tyThe nanostructured lipid carrier in this experiment was considered to be the reason for the superior effect of the DHA-loaded NLC group compared with DHA alone. Meanwhile, the minimal anti-inflammatory and osteoprotective effects of the NLC group were attributed to the liquid lipid squalene in the nanostructured lipid carrier. The creation of multifunctional nanoparticles with anti-inflammatory properties using the natural lipid squalene and the endogenous immunomodulator adenosine managed endotoxemia in rodents . This isComprehensively, this study innovatively prepared DHA-loaded NLCs by compounding squalene as an antioxidant with DHA, giving full play to its structural advantages, enabling slow local release and providing high concentrations of the drug to exert anti-inflammatory effects with fewer side effects and higher safety. This innovative form of DHA overcame the drawbacks of DHA, including quick oxidation and insolubility in water. Additionally, DHA-loaded NLCs may transform the administration form of DHA from oral medication to local administration, opening a new avenue for the treatment of peri-implantitis and providing a new technique for overcoming complex and multifactorial inflammatory phenomena.Nonetheless, there are some limitations of this experiment based on the aforementioned studies. For instance, DHA-loaded NLCs were injected into the inflammatory gingival tissue; therefore, the injection dose was extremely limited. Moreover, this manner of administration would cause some discomfort and even pain, which needs further optimization and improvement of the dosage form. In addition, large animals, such as domestic pigs, should be furtherly employed in animal experiments instead of rodents to better inform clinical practice. DHA-loaded NLCs were fabricated through ultrasonication using 800 mg of monostearin , 200 mg of squalene , 500 mg of DHA , 270 mg of Tween 80 , 30 mg of glycerol , and 7200 mg of ultrapure water. Briefly, ultra-pure water was added to an oil phase composed of two lipids , DHA, and two surfactants (Tween 80 and glycerol). Then, the reagents were heated together at 70 °C to promote binding. Subsequently, the liquid was agitated for 30 min at 500 rpm  to create a milky pre-emulsion. The DHA-loaded NLCs (DHA-NLC) were then obtained by using a sonicator  at 60% amplitude for 5 min.The morphology of the DHA-loaded NLCs was observed using transmission electron microscopy . The sample was obtained by dropping a small amount of the formulation onto a 100-mesh copper mesh and then diluting it 50 times with 3% phosphotungstic acid for negative staining.A Zetasizer Nano-ZS90 was used to measure the particle size, polydispersity index (PDI), and zeta potential of the DHA-loaded NLCs . The test samples were diluted 5 times with deionized water at 25 °C. All samples were tested three times.UV/VIS-spectroscopy  was used to quantify the DHA nanoencapsulation. DHA-loaded NLCs were dissolved in n-hexane for 30 min, centrifuged for 30 min at 12,000 rpm, and then evaluated by measuring the absorbance of the supernatant at 272 nm using a UV/VIS spectrophotometer. By determining the amount of free DHA in the DHA-loaded NLC dispersions using the following equations, EE and LC were indirectly calculated:−1 in 64 scans, FTIR spectroscopy of the DHA, blank-NLCs, and DHA-loaded NLCs was conducted. The sample was combined with potassium bromide powder  and a thin plate of potassium bromide served as the background. A pellet of the combination was formed and measured by FTIR in the 400–4000 cm−1 range.Using an FTIR spectrometer  with a resolution of 4 cmWith a few minor alterations, this experiment was based on Qayyum Shehzad’s methodology . The abiThe release profile of DHA from the DHA-loaded NLCs was assessed using a dynamic dialysis technique. Then, 2 mL of DHA-loaded NLCs was added to a dialysis bag. The dialysis bag was then placed in a centrifuge tube with 40 mL of PBS solution and 1% Tween 80 as the dialysis medium. In a thermostatic gas bath, the centrifuge tube was maintained at 37 °C and 150 rpm. One milliliter of the release solution was swapped out for the same amount of dialysis medium at predefined intervals. A UV/VIS spectrophotometer was used to assess the absorption at 276 nm.The drug was stored in a refrigerator at 4 °C and the surface charge and dimensions were periodically measured to assess its stability.2 in a humidified cell incubator. Cultured cells that were 70% confluent were used. Afterward, 96-well plates were inoculated with 1 × 104 macrophages per well and cultured for 12 h. Following 2 h of pretreatment with each drug group, the cells were stimulated with P. gingivalis LPS  for 24 h. (The drug concentration was based on the concentration of DHA in the DHA-loaded NLCs. The concentrations of the DHA solution and NLC should be consistent with those of DHA-loaded NLC.) Cell Counting Kit-8  solution (10 μL) was added to the wells and incubated for 2 h at 37 °C. A microplate reader  was used to determine the absorbance at 450 nm.Macrophages were provided by the Shanghai Institute of Cellular Sciences, China. Briefly, macrophages were cultured in Dulbecco’s Modified Eagle Medium  with 10% fetal bovine serum  and 1% penicillin–streptomycin solution at 37 °C with 5% CO5 cells/well, and 50 μM was set as the final concentration in the formal experiment by screening in the pre-experiment. Trizol reagent  was used to extract the total RNA, and Hifair® III 1st Strand cDNA Synthesis SuperMix for quantitative polymerase chain reaction (qPCR) (gDNA Digester+)  was used to synthesize cDNA for the Roche LightCycler® 96 RT-qPCR platform’s real-time polymerase chain reaction (PCR) . The primer sequences used for qRT-PCR are listed in TNF-α, IL-1β, and IL-6 as the specific primers and β-actin mRNA as an internal reference.The procedure for macrophage cultivation was the same as that mentioned above. The cell inoculation density was 2 × 104 cells per well for 24 h. Then, the macrophages were stimulated with LPS (5 μg/mL) for 3 h. The cells were collected, fixed, and permeabilized sequentially. Thereafter, the cells were incubated with nuclear factor-κB (NF-κB) p65  overnight at 4 °C and then rabbit Alexa Fluor 455 anti-mouse antibody  for 1 h on the following day at room temperature. The number of NF-κB p65-positive cells was observed and measured under an Olympus microscope.As previously mentioned, the macrophages were cultured. Drugs were co-cultured with 1.5 × 10Male adult Wistar rats  were kept at room temperature (22 °C ± 2 °C) with a 12:12 light/dark cycle and had free access to food and water. The First Hospital of Jilin University’s Animal Ethics Committee approved the use of animals in this experiment (No. 2022-0576).P. gingivalis LPS was injected into the peri-implant fissure every 3 days in the following 2 weeks to establish peri-implantitis. The peri-implant gingival tissue was then immediately injected with 100 μL of each group emulsion using an insulin syringe  at the four points of the implant  every 3 days within 2 weeks. Afterward, 4% paraformaldehyde was used to fix the dissected rat maxillae before performing microcomputed tomography (micro-CT) and hematoxylin and eosin (H&E) staining. For qRT-PCR, the peri-implant gingival tissue was extracted. The centrifuged serum and right ventricle blood samples were used to evaluate liver function and perform regular blood tests. Hearts, livers, spleens, and kidneys were also harvested for in vivo toxicity testing.After sequential cleaning, surface sandblasting, large-grit etching, and acid etching (SLA), titanium SLA mini-implants  were obtained  . The ratMicro-CT  was used to analyze the bone tissue. The scanning parameters were as follows: voxel size of 34.4 μm, 200 mA, 70 kVp, and exposure time of 300 ms. The trabecular thickness  and bone volume fraction  within the regions of interest were analyzed.Magnetic beads were used to grind the peri-implant gingival tissue, and TRIeasy was used to extract the mRNA. The synthesis of cDNA and qRT-PCR was performed referring to the methods mentioned above. To compare the relative mRNA expression levels, the in vivo qRT-PCR primers are listed in After rinsing under flowing water and fixing with paraformaldehyde, the maxillae were decalcified with a 10% EDTA solution at 4 °C for 2 months. The EDTA solution was refreshed every 3 days. The implants were removed using an implanted spanner once the decalcification process was finished. Then, 2 μm tissue sections were stained with H&E and the amount of inflammatory cell infiltration was measured to determine the degree of inflammation in the gingival tissue surrounding the implants.Blood tests, albumin, alanine aminotransferase, and aspartate aminotransferase analyses were performed. Following fixation, histopathological analysis was used to evaluate the drug toxicity in the primary organs of rats.p-value of <0.05 was considered statistically significant. There were three iterations of each experiment in this study.All experimental results were statistically analyzed using Prism 8.0 , and they were presented as the mean ± standard deviation (SD). One-way analysis of variance was used to evaluate the differences between groups, and a DHA-loaded NLCs overcome the drawbacks of DHA, including quick oxidation and insolubility in water. Additionally, DHA-loaded NLCs represent a unique approach to intercept the pathogenicity of oxidative stress to inflammation through suppressing the NF-κB pathway. Local delivery of DHA-loaded NLCs provides a novel strategy for the application of DHA for the treatment of peri-implantitis, as well as other oxidative stress-related inflammatory diseases, in clinical practice.


Inoculation theory has become an increasingly common framework for reducing susceptibility to both individual examples of misinformation (issue-based inoculation) and to the techniques and strategies that are commonly used to mislead or misinform people (technique-based inoculation). In this study, we address two open questions related to technique-based inoculation in two separate experiments (total  Durinpassive inoculation, or it actively involves the participant in the generation of those counterarguments, known as active inoculation , but not real news [1bH] as significantly less reliable after playing; and2:H people who play Bad News improve in their ability to discern the reliability of real-life examples of manipulative and non-manipulative news.The misinformation items were a combination of previously used headlines by Traberg & van der Linden  and new ironment . For the. 3.23 with 58.0% having obtained a higher degree. Participants were also somewhat left-leaning . See the electronic supplementary material, table S1 for the sample composition.A total of 1 216 valid responses were collected. The sample consists of 55.1% men, with 48.0% of survey participants reporting being between 18 and 29 years old,. 3.3t-tests on the pre- and post-scores for each social media post (item), as well as on the aggregated scores for the misinformation and real news (control) items . For the misinformation items , we find a significant reduction in perceived reliability post-gameplay  ). This translates to 64.4% of the post-gameplay reliability scores being lower than the mean of pre-gameplay reliability scores (i.e. Cohen's U3 = 64.4). We also find that participants rate all six misinformation posts that made use of one of the manipulation techniques learned in Bad News as significantly less reliable after playing . In addition, participants rated the post about coronavirus as significantly less reliable after gameplay , albeit with a substantially lower effect size. We note that the pre-score for the coronavirus post is the lowest out of all the items and is even lower than the lowest post-gameplay score for the other items , indicating possible flooring effects .Following Roozenbeek & van der Linden , we condF2,2426 = 64.8, p < 0.001, η2 = 0.025), a finding consistent with results from Roozenbeek & van der Linden  and item set B–B [1bH];2:H when comparing an index of the same items, pre-test (group x) with post-test (group y), there is no significant decrease in the perceived accuracy of real headlines, both for item set A-A [2aH] and item set B–B [2bH];3:H when comparing different item sets, pre-test (group x) with post-test (group x), there is a significant decrease in the perceived accuracy of false headlines, both for group A-B [3aH] and group B-A [3bH];4:H when comparing different item sets, pre-test (group x) with post-test (group x), there is no significant difference in the perceived accuracy of real headlines, both for group A-B [4aH] and group B-A [4bH]; and5:H people who play Bad News significantly improve in their ability to discern the accuracy of real-life examples of real and false headlines.Again following Roozenbeek et al. , we test. 4.2M = 3.17, s.d. = 1.39). In addition, we also asked participants about their region of origin , and whether participants had played Bad News before . See the electronic supplementary material, table S1 for the full sample composition.A total of 968 valid pre-post survey responses were collected from the in-game survey. The sample consisted of 50.7% men, with 55.5% of respondents being between 18 and 29 years old, and 51.8% having obtained a higher degree. As in experiment 1, participants were left-leaning (. 4.31H and 2H), comparing item set A (B) pre-test scores with item set A (B) post-test scores. Doing so yields a significant effect for the false news items for item set A (pre-test for the A-B group) versus item set A (post-test for the B-A group) . These results support hypothesis 1aH. However, we find no significant difference for the false news items for item set B (pre-test for the B-A group) versus item set B (post-test for the A-B group) ; see the electronic supplementary material, tables S8 and S9. To check whether this result implies the absence of an effect of interest, we performed a two one-sided t-test (TOST) equivalence test with a smallest effect size of interest of d = (−)0.20 and α = 0.01 [5 Doing so confirms statistical equivalence to zero: t964 = −2.60, p = 0.005. Our data thus does not support hypothesis 1bH.We first look at whether an inoculation effect is found when crossing the item sets between groups  and item set B ; see the electronic supplementary material, tables S8 and S9. However, A TOST equivalence test does not confirm statistical equivalence to zero for item set A  or item set B , which means that we cannot rule out that there is an effect  that is larger than d = 0.20. These results support hypothesis 2aH, and partially support hypothesis 2bH.We perform the same analyses for the real news items. Doing so yields no significant difference for both item set A (3H and 4H). When looking at groups A-B and B-A together, we find a significant overall reduction in perceived accuracy post-gameplay for the false news items . This means that 54.0% of the post-gameplay false headline accuracy scores are lower than the mean of pre-gameplay accuracy scores (i.e. Cohen's U3 = 54.0). However, this effect is only significant for the A-B group , but not for the B-A group ; see the electronic supplementary material, tables S8 and S9. A TOST equivalence test confirms statistical equivalence to zero for the B-A group: t454 = 3.31, p < 0.001. We thus find support for hypothesis 3aH, but not for hypothesis 3bH.Next, we examine whether inoculation effects can be detected when comparing different item sets within the same groups . However, for the A-B group, we find a significant decrease in perceived reliability of real news , whereas for the B-A group, we find the opposite, namely a significant and descriptively larger increase in the perceived reliability of real news ; see the electronic supplementary material, tables S8 and S9. We thus find no support for either hypothesis 4aH or hypothesis 4bH.For the real news items, we find no significant overall difference in perceived reliability post-gameplay . These results support hypothesis 5H; see the electronic supplementary material, tables S8 and S9. In terms of participants' ability to distinguish real and false news headlines, we find a significant overall increase in truth discernment post-gameplay, indicating that p's > 0.063). Second, we conduct a linear regression with the same factor and independent variables, but with the difference in truth discernment before and after gameplay as the dependent variable. Doing so shows that none of the covariates predict the pre-post-inoculation effect for truth discernment , except age (p = 0.005), so that being older is associated with a higher pre-post difference in truth discernment; see the electronic supplementary material, table S10.Finally, as in experiment 1, we check whether any covariates influence the observed inoculation effects. To do so, we again first conduct a linear regression with the average pre-post different scores for the misinformation  items as the dependent variable, group (A-B or B-A) as a factor variable, and gender, age, education and political affiliation as independent variables. Doing so shows that none of our covariates significantly predicts the pre-post-inoculation effect for false headlines  inoculation effect even for misinformation that Bad News players were not inoculated against, indicating partial cross-protection [Overall, we find that playing the  A and B . This isotection .. 5Bad News game confers psychological resistance against misinformation techniques used in real-life examples of online misinformation. In line with previous research, we find that players' truth discernment, or their ability to distinguish manipulative from non-manipulative information, significantly improves after gameplay. Importantly, this effect is primarily driven by a decreased perceived reliability (or accuracy) of misinformation. For real news, the findings are somewhat more ambiguous; while in experiment 1, we find a small but significant decrease in perceived reliability, we find the same effect for one item set used in experiment 2, but the opposite in the second item set, namely an increase in the perceived accuracy of real news , the observed decrease in the perceived reliability of real news warrants further discussion, as this is not an explicit goal of the inoculation intervention. A similar phenomenon was observed for a different anti-misinformation intervention, so-called ‘digital literacy tips' . We arguWith respect to false news, while our findings in experiment 1 are straightforward, the results from experiment 2 are more ambiguous: effect sizes reported in this experiment are descriptively smaller than in experiment 1, and group B-A showed no significant decrease in the perceived accuracy of false headlines . That saDEPICT misinformation techniques learned while playing Bad News , and players were not specifically inoculated against these types of false headlines . This may explain the differences between the present study and those reported in Roozenbeek et al. [Bad News game is at least partially transferrable to previously unseen types of misinformation. In other words, active inoculation treatments appear to be at least somewhat effective at conferring cross-protection against related but untreated persuasive attacks [same misinformation technique) versus true cross-protection, which could occur when resistance is formed against novel misinformation that either does not make use of the same strategies (as in the current study) or makes more explicit use of entirely different strategies (akin to different topics) that people were not inoculated against. Future research will have to examine—in a more controlled way—whether inoculation games can also confer cross-protection in the sense that inoculating people against one misinformation technique (e.g. conspiracy theories) also confers protection against unmentioned and entirely different techniques (e.g. impersonation).One reason for this non-predicted effect may be that the false headlines we used in experiment 2 did not make use of the k et al. , who did attacks ,15,28. TAlthough we conducted two studies, we were still unable to account for several key limitations. First, with respect to covariates, although we do not find political ideology to be an independent predictor of the inoculation effect , previous research has found political similarity with sources to impact misinformation susceptibility . As sucha priori, and we note the presence of item effects that may complicate our findings. We were only able to use a limited number of items in each experiment, and the low internal consistency for our item sets, especially in experiment 2, limits the generalizability of our findings. Third, this study also explored whether eliciting different question framings (e.g. the reliability or accuracy of an item set) can be expected to yield different response patterns when testing the efficacy of anti-misinformation interventions. Our results are inconclusive: although we find that both question framings yield significant results in the hypothesized direction, the effect sizes in experiments 1 and 2 are substantially different. However, because we used different item sets, we are unable to directly compare both experiments' response patterns. Thus, while this study is to our knowledge, the first to test two separate outcome measures (reliability and accuracy) on the same intervention, it is unclear to what extent these measures are interpreted differently by participants, and how this may affect their item ratings; see Roozenbeek et al. [Second, our item sets were not psychometrically validated k et al. .Finally, more can be said about the mechanisms underlying the efficacy of active inoculation games. In related research, scholars have provided some early evidence for the role of motivational threat , which pBad News improves people's ability to spot misinformation [Overall, we corroborate previous findings that playing ormation ,30,32. Tormation , ‘prebunormation , accuracormation ,47 or me


WT) and POX knockdown MCF-7 cells (MCF-7crPOX cells) was studied. PRODH/POX catalyzes proline degradation generating ROS-induced apoptosis or autophagy. Availability of proline for PRODH/POX functions is regulated by the activity of prolidase (enzyme releasing proline from imidodipeptides), collagen biosynthesis (process consuming proline), and metabolism of proline, ornithine, and glutamic acid. We have found that MET is cytotoxic for MCF-7 cells (IC50∼17 mM), and to the lower extent for MCF-7crPOX cells (IC50∼28 mM). In MCF-7WT cells, the effect was accompanied by the inhibition of DNA biosynthesis, collagen biosynthesis, stimulation of ROS formation, AMPKα phosphorylation, and expression of prolidase, p53, caspase 8, caspase 9, and cleaved PARP. In MET-treated MCF-7crPOX cells, the processes were less affected than in MCF-7WT cells and the expression of caspase 9 was decreased, while cleaved caspase 8 and cleaved PARP were not detected. The effects were accompanied by an increase in the prolidase activity and proline concentration. The mechanism for MET-induced apoptosis involves the up-regulation of prolidase activity and a decrease in collagen biosynthesis contributing to an increase in the concentration of substrate (proline) for PRODH/POX-dependent ROS formation and activation of caspases −9 and −8. The data suggest that PRODH/POX participates in the MET-induced intrinsic and extrinsic apoptosis in MCF-7 cells.Although the antineoplastic activity of metformin (MET) is well established, the underlying mechanism of the activity is not understood. Since MET activates AMP kinase (AMPK) and proline dehydrogenase/proline oxidase (PRODH/POX) is stimulated by AMPK ligands , the effect of MET on PRODH/POX-dependent apoptosis in wild-type MCF-7 cells (MCF-7 Metformin (MET) is widely used to treat type II diabetes. It evokes multidirectional activity by a decrease in the intestinal glucose absorption, the inhibition of gluconeogenesis, an increase in glycolysis, tissue sensitivity to insulin, and hypoglycemia . HoweverActivation of AMPK through its phosphorylation takes place in conditions of energy shortage  when the ratio of AMP/ATP increases. This kinase, in order to restore the proper level of ATP, stimulates oxidative phosphorylation and inhibits the expenditure of energy, e.g., for cell proliferation . The ovePRODH/POX catalyzes the conversion of proline to ∆1-pyrroline-5-carboxylate (P5C). This process yields electrons that could be transported to the electron transport chain producing ATP-promoting cell survival  or they Another factor regulating the intracellular concentration of proline is collagen biosynthesis . SynthesIt has been suggested that the PRODH/POX-dependent generation of ROS may contribute to the initiation of intrinsic and extrinsic apoptotic pathways . PRODH/PHowever, PRODH/POX-mediated apoptosis could occur through different mechanisms in different cancer cells. It may involve the deregulation of cell signaling and cell cycle regulatory pathways. One of the most potent PRODH/POX activators is protein p53. PRODH/POX could be therefore transcriptionally regulated by p53 since the promoter of PRODH/POX contains a specific p53-response element .crPOX cells).The aim of this study is the identification of the mechanism of anticancer activity of MET in breast cancer cells. We evaluated the effect of MET on the expression of AMPK, PRODH/POX, cytoplasmic concentration of proline, collagen biosynthesis, and some apoptosis-inducing proteins in MCF-7 and PRODH/POX knockdown MCF-7 cells (MCF-7Escherichia coli DH5α cultured in Luria-Bertani (LB) medium with 100 μg/ml ampicillin for 24 h at room temperature. The extraction of the targeted plasmid was performed using a plasmid DNA purification kit . The purified samples were precipitated with isopropanol, washed with 70% ethanol, and submitted to DNA cleaning-up step by the GeneMATRIX Basic DNA Purification Kit . DNA concentration was evaluated by NanoDrop™ 2000/2000c Spectrophotometer.The sgRNAs for PRODH/POX  were products of ABM Company. The vector containing expression construct was transfected into 2. The cells were cultured in 6-well plates to achieve about 70–90% of confluency. The amount of plasmid tested in the experiment was 1–2 µg/well. For transfection, lipofectamine 2000 (Invitrogen) was used.MCF-7 cells were cultured in DMEM 1X (Gibco), containing 10% fetal bovine serum (FBS) qualified (Gibco), 4.5 g/L glucose, L-glutamine, and pyruvate supplemented with 1% penicillin/streptomycin (Invivogen) at 37°C in 5% CO2. The next day, the transfected cells were selected in the complete growth medium with 1 μg/ml of puromycin (Sigma-Aldrich) in the same culture conditions for 10 days. The PRODH/POX expression was analyzed by Western blot. Based on the expression level, the PRODH/POX knockout MCF-7 cell line was selected for further stable clone generation. The screening steps were done with a random selection of cell clones. The efficiency of PRODH/POX silencing was measured by Western blot using an anti-PRODH/POX antibody (Santa Cruz). The PRODH/POX knockout MCF-7 cells defined as MCF-7crPOX cells were banked for further experiments.Prior to transfection, the plasmid was diluted with 50 µL of medium A, DMEM 1X (Gibco) without FBS. The transfection solution (805.4 µL of medium A and 194.6 µL of lipofectamine) was gently mixed and incubated for 5 min at room temperature. The cells tested were washed with PBS 1X , 1 ml of medium A was added and incubated for 20 min, and then the mixture of plasmid with transfection reagent was added to cells and incubated overnight at 37°C in 5% COWT) and MCF-7crPOX cells were cultured in DMEM with 10% fetal bovine serum, 50 IU/ml penicillin, and 50 μg/ml streptomycin at 37°C in 5% CO2. The medium was changed every 3 days until the cells reached 80% of confluency. Then, the cells were treated for 48 h with MET at a concentration of 0–80 mM in glutamine-free medium.The MCF-7 breast cancer cells (HTB-22) were purchased from ATCC Company . Wild-type MCF-7 cells  incorporation into proteins susceptible to Clostridium histolyticum collagenase, as described previously  in the positive ionization mode according to the method of Klupczynska et al. . ChromatAccumulation of ROS was assessed by using DCFH-DA (fluorescent probe), as described previously . The celThe translocation of phosphatidylserine (PS) to the cell surface due to MET treatment was measured using the Muse® Annexin V and Dead Cell Kit . The combination of FITC-labeled Annexin V with 7-AAD  allowed us to distinguish the apoptotic cells. For the purpose of the clarity of presented data, live cells (Annexin V-negative and 7-AAD-negative) were not presented in the provided figures.Immunostaining was conducted according to the BDB Bioimaging protocol, as described previously . Cells exposed to MET (20 mM) were fixed with paraformaldehyde, permeabilized with Triton, blocked with 3% FBS, and incubated with anti-cleaved-caspase-7 (primary antibodies) and FITC Fluor-conjugated secondary antibody and Hoechst. Samples were visualized with a confocal laser scanning microscope (BD Pathway 855 Bioimager) using AttoVision software.p < 0.01 and **p < 0.001.All experiments were carried out in duplicates, and the experiments were repeated at least three times. Data are shown as a mean ± standard deviation (SD). For statistical calculations, a one-way analysis of variance (ANOVA) with Dunnett’s correction and t-test was used. Statistical analysis was performed using GraphPad Prism 5.01 . Statistically significant differences were marked as *WT), expressing PRODH/POX and on PRODH/POX knocked out cells (MCF-7crPOX) generated by the CRISPR/Cas9 method. The effect of MET on the cell viability in both cell lines was measured by the MTT assay , a fluorogenic dye that when oxidized by ROS produces fluorescent dichlorofluorescein (DCF). Cells were treated with 20 mM of MET, a concentration that decreased the viability of MCF-7 control . It suggWT by about 50%, while in MCF-7crPOX, it increased only by about 25%, compared to non-treated cells  protein expression due to MET-treatment. In MCF-7crPOX cells, the phenomenon was not observed.MET has been identified as a potent stimulator of AMP-dependent kinase (AMPK). The enzyme is a heterotrimer composed of alpha, beta, and gamma subunits. The alpha subunit is post-translationally activated by phosphorylation. In both cell lines treated with MET, an increase in phosphorylation of the WT cells. Changes in the expression of initiator caspases are accompanied by the cleavage of PARP—one of the caspase 3 substrates. In both cell lines, MET induced the expression of PARP; however, the cleaved PARP was found only in MCF-7WT cells treated with 10 and 20 mM MET. The observed changes in the protein expression confirm the activation of apoptosis in MCF-7WT cells treated with MET. The results were confirmed by immunofluorescence bioimaging of caspase-7 in studied cells (The p53 protein is a key factor in activating apoptosis in response to DNA damage. MET-induced increase in the p53 expression in both cell lines was accompanied by a decrease in the expression of procaspase-9 and the absence of cleaved caspase-8. Expression of caspase 8 was induced by 20 mM MET only in MCF-7ed cells .WT and MCF-7crPOX). The biosynthesis of collagen requires a large supply of proline; therefore, when the process is reduced, it supports free proline for PRODH/POX-dependent functions. At 20 mM, MET induced the expression of prolidase, an enzyme catalyzing the recovery of proline from imidodipeptides (degradation products of collagen) in MCF-7WT cells. The process was less pronounced in MCF-7crPOX cells. An increase in the prolidase expression should result in the increase in the intracellular proline concentration, thus providing a substrate for PRODH/POX. These MET-dependent processes were accompanied by a dose-dependent decrease in the expression of ERK1/2 in both cell lines.As presented in WT cells and to a lesser extent in MCF-7crPOX cells  contributed to an increase in the concentration of proline in the cells  and strongly induced apoptosis only in MCF-7WT cells. It seems that the mechanism of MET-induced apoptosis could be attributed to the inhibition of collagen biosynthesis and an increase in proline availability for PRODH/POX and ROS generation. In this report, we have provided evidence that the treatment of MCF-7WT cells with MET increased ROS formation and the expression of caspase 8, caspase 9, and cleaved PARP. In MET-treated MCF-7crPOX cells, the processes were less affected.In this report, we provide evidence for the important role of PRODH/POX in the mechanism of MET-induced apoptosis in MCF-7 cells. We hypothesized that the sequence of events could involve i/MET-induced AMPK, ii/AMPK-induced PRODH/POX, and iii/PRODH/POX-dependent ROS generation inducing apoptosis. The hypothesis was based on previous reports showing that the overexpression of PRODH/POX contributed to apoptosis by intrinsic and extrinsic pathways . HoweverThe data suggest that both PRODH/POX and proline availability for the enzyme play an important role in the mechanism of MET-dependent apoptosis in MCF-7 cells. Since PRODH/POX is considered as an inhibitory factor in tumor progression , the metin vivo conditions, proline has an advantage over fatty acids, glutamine, and glucose that require delivery by the circulation. In the presented conditions, proline could be directly used from extracellular matrix protein degradation. In this context, proline may serve as an energy substrate as well as an energetic sensor. In fact, in conditions of energy shortage, the cells adapt to the metabolic changes by activating AMPK  participating in proline shuttling between mitochondria and cytoplasm. The conversion of P5C into proline is linked to glucose metabolism by the pentose phosphate pathway, while the conversion of proline into P5C is coupled to the TCA cycle . It is vHowever, in several of our previous studies, we found that the role of PRODH/POX in the apoptosis/survival of cancer cells depends on the metabolic context of a specific cell type. In one of our recent articles , we provIt has been considered that proline availability for PRODH/POX could be an important factor in the creation of pro-apoptotic phenotype of cancer cells. In melanoma cells, characterized by the intense biosynthesis of collagen (proline consuming process) PRODH/POX knockout did not induce apoptosis. It seems that in the melanoma cells with a high capacity to utilize proline for collagen biosynthesis, the proline concentration is not high enough to induce extrinsic apoptosis. However, the stimulation of PRODH/POX by MET in melanoma cells induced ROS-dependent apoptosis, while PRODH/POX knockout abolished the effect . A similTherefore, we suggest that the role of PRODH/POX in apoptosis/survival in cancer cells is metabolic context-dependent and the process occurs through various mechanisms in different types of cancer cells. Obviously, it limits the clinical relevance of MET use in breast cancer. To date, a decreased risk of breast cancer was observed only in patients with type 2 diabetes, receiving MET on a long-term basis . Similarin vitro studies that should be confirmed by in vivo experiments. Although cell line models have several limitations , they are a powerful tool that offers several advantages. Certainly, the cell models allow a strict control of the conditions of the experiment in order to establish the critical factor affecting the studied processes in a relatively short time. They are especially helpful in the case of limited availability of clinical samples or in vivo models. Therefore, results on cell models allow us to predict the consequences of pharmacotherapeutic manipulation in humans and provide rational for clinical studies on dose-dependent effects. Different treatment regimens and combinations of therapies have been tested using cell lines that have yielded interesting and potentially promising results that some of them could have an application value.The application value of the results presented in this article has obvious limitations due to In this report, we suggest that the mechanism for MET-induced apoptosis involves an increase in the prolidase activity and a decrease in collagen biosynthesis providing substrate (proline) for PRODH/POX-dependent ROS formation and activation of caspases-9 and -8. The data suggest that PRODH/POX participates in the MET-induced intrinsic and extrinsic apoptosis in MCF-7 cells.MET-induced apoptosis involves the up-regulation of PRODH/POX-dependent ROS formation, the activation of caspases-9 and -8, an increase in prolidase expression and activity, a decrease in collagen biosynthesis, and an increase in the proline concentration providing a substrate for PRODH/POX. The data suggest that PRODH/POX participates in the MET-induced intrinsic and extrinsic apoptosis in MCF-7 cells.